We continue to work toward a complete understanding of the interaction of melanotropin (MSH) with its receptors and the way in which the proliferation and pigmentation of normal and malignant pigment cells are controlled. In addition, we want to characterize the oncogenes for melanoma and their gene products. Our procedure for preparing 125I-beta-MSH is now standardized, and we have optimized the conditions for an assay to measure the binding of the ligand to Cloudman S-91 melanoma cells. This achievement will serve as a foundation from which to characterize more fully the receptor for beta-MSH on Cloudman S-91 melanoma and other MSH-responsive cells. The mechanism of action of MSH is being studied by different labeling techniques. The procedures involve the use of 125I-beta-MSH, Ferritin-FITC-MSH, and covalent labeling of the receptor. We have raised antisera to MSH from three rabbits and hope to identify MSH bound to the receptor immunocytochemically, both at the cell surface and within cells at sites of synthesis, processing, degradation, and possibly action. We have improved the method of growing pure cultures of normal human melanocytes in the presence of TPA (4-O-methyl-12-O-tetradecanoyl-phorbol-13-acetate) and geneticin and have also developed a method of growing pure cultures of avian melanocytes from neural crest. From these two sources we are currently growing normal melanocytes in large quantities (1 x 109 cells) and are able to use these cells for biochemical studies. Ongoing experiments designed to identify the malignant process in melanomas involve identifying the gene products responsive to TPA and cholera toxin. We have identified three TPA-responsive proteins in normal human melanocytes whose functions are expressed constitutively by human melanoma cell lines. (B)